The low-density lipoprotein receptor (LDL-R) is a member of an array of membrane-associated receptors that collectively bind more than 40 ligands. The human LDL-R avidly binds to apolipoproteins B and E, which carry the majority of circulating cholesterol to the liver, arterial wall, brain and steroidogenic organs. The classic LDL-R regulatory model entails active upregulation by low sterol by way of a sterol response- element binding protein (SREBP). Basal gene expression is therein viewed as a passive process of nonstimulation. In contrast, based upon pilot data, we propose a paradigmatic shift in the concept of LDL-R gene regulation, wherein transcriptional proteins in the superfamily of Krueppel-like factors (KLF's) actively silence basal LDL-R gene transcription. One of the factors, KLF13, can both repress and activate the pig LDL-R promoter in swine ovarian and insect SL2 cells. The same protein upregulates the erythroid globin, lymphocyte RANTES and SM22-alpha, and downregulates the CYP1A1 and KLF13 promoters. These data frame the novel thesis that KLF13 serves as a molecular switch between active LDL-R gene repression and derepression. The exploratory and developmental nature of the R21 is to test this unique model in liver and vascular smooth muscle cells (VSMCs), which mediate large fluxes in LDL cholesterol. The significance of understanding the role of KLF13 arises in targeting the activity of this transcriptional peptide pharmacologically, so as to upregulate LDL-R gene expression in the liver and downregulate the same in VSM, thereby in principle interrupting the atherogenic cycle at two key loci of control. To this end, we identify 2 Specific Aims stated here as Hypotheses: Aim I. KLF13 actively inhibits basal LDL-R gene expression; and Aim II. Activation of the LDL-R promoter by low sterol requires reversal of gene silencing. Technical strategies to address these issues include high-efficiency viral transfection of hepG2 and vascular smooth muscle (VSM) cells; RNA interference to knock down transcriptional-factor messages; chromatin immunoprecipitation (ChIP) of LDL-R promoter-bound proteins; and SL2 insect-cell expression of proposed coregulators. In summary, this R21 proposal is designed to test a novel paradigm of LDL-R gene regulation, wherein strong repression of basal transcription must be overcome to allow promoter activation. Outcomes have the potential to unmask a new interventional strategy to unleash LDL-R expression in the liver and silence LDL- R expression in the arterial wall. Layman's summary Atherosclerosis is driven in part by excessive cholesterol deposition in the arterial wall and reciprocal failure of cholesterol removal by the liver. We have identified a gene in ovarian cells that, unexpectedly, can both increase and decrease cellular uptake of cholesterol-laden LDL particles. This project tests whether the product of this gene could decrease LDL deposition in arterial muscle and increase LDL removal by the liver. If so, targeting this gene product could potentially shift cholesterol accumulation away from arteries toward the liver. This would represent a new treatment approach for patients with atherosclerotic disease. [unreadable] [unreadable] [unreadable] [unreadable]